We propose an innovative structural biology approach to stimulate pre-erythrocytic malaria vaccine and drug development using integrated information from X-ray crystallography and cryoelectron tomography to develop a detailed molecular topography of the sporozoite surface. This work will provide a three-dimensional map of the native arrangement of two of the most critical surface proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP), and their most potent epitopes on the sporozoite surface. This work will also be of significance for providing insights into the structural mechanisms of parasite motility, cell invasion, and immune evasion. With the long-term goal of stimulating rational malaria vaccine development and structure-based drug design, we have three specific aims. 1. We propose that CSP packs in a specific way to form the sporozoite sheath. CSP contains N-terminal, repeat, and thrombospondin type I repeat (TSR) domains. We will complete work on the crystal structure of the CSP TSR domain, which we call an -TSR domain because it contains a unique N-terminal -helix and other unique features compared to previously solved TSR domains. Surprisingly, -TSR packs as a trimer in different crystal lattices with the -helix central in the trimer. The shed CSP and isolated -TSR domain are monomers at ~ mg/ml concentrations. We hypothesize that the -TSR domain is a trimer on the parasite surface and packs in a hexagonal lattice to form a sheath, and that the trimer to monomer conversion upon shedding is part of a shape-shifting strategy for immune evasion. We will stabilize the trimeric state and test the hypothesis that antibodies to it more effectively blck infection in vivo and liver cell invasion in vitro than antibodies to the monomer. 2. We will solve the crystal structure of the tandem von Willebrand factor A (VWA) and TSR domains in TRAP. We wish to understand how ligand binding to TRAP mediates gliding motility and cell invasion. Structures will test the hypothesis that the VWA and TSR domains interact with one another in a manner conducive to conformational change transmitted by tensile force between the VWA domain and the cytoplasmic domain. 3. We will apply cryoelectron tomography to define ultrastructural details of the sporozoite surface. We expect to see three sheath layers corresponding to the N-terminal, repeat, and TSR domains, that differ in electron density and in packing. We expect to be able to use subtomogram averaging to analyze packing in the most membrane- proximal layer, and to compare this packing to that of the -TSR domain trimer crystal lattices. We may also see islands of other molecules such as TRAP in a sea of CSP. Through complementary use of crystallography and cryoelectron tomography and microscopy we plan to construct a model of the sporozoite sheath. These findings should provide important insights into how epitopes are shielded or exposed in the sheath at different times in the infection process, and how apicomplexans shield themselves from the immune system. PUBLIC HEALTH RELEVANCE: Malaria affects 250 million people world-wide. We will determine key surface structures of the parasite, to advance vaccine development.